2 palmitoylglycerol 2 pg

Lipids were extracted from mouse livers as described earlier [11 (link), 28 ]. Briefly, frozen mouse liver (100–200 mg wet weight) was homogenized in 1.0 mL of ice-cold homogenization buffer containing each of the deuterated standards AEA-d₄, OEA-d₂, PEA-d₄, DHEA-d₄, EPEA-d₄, and 2-AG-d₈ such that the final amount of each standard was 2000pg. The final lipid residue was redissolved in 100 μL of ice-cold methanol, purged with N₂, and stored at −80°C until liquid chromatography-mass spectrometry (LC-MS) analysis below.
LC/MS analysis of liver NAE and 2-MG was performed as described earlier by our lab [30 (link), 31 (link)]. Briefly, identification and quantitation of individual NAE or 2-MG was accomplished through use of: i) Addition of internal standards to each aliquot of liver homogenate including deuterated AEA-d₄, OEA-d₂, PEA-d₄, DHEA-d₄, EPEA-d₄, and 2-AG-d₈ from Cayman Chemical (Ann Arbor, MI); ii) Comparison with external standard curves of n-6 arachidonoylethanolamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA), n-3 docosahexaenoylethanolamide (DHEA), n-3 eicosapentaenoylethanolamide (EPEA), 2-arachidonoylglycerol (2-AG), 2-oleoylglycerol (2-OG), and 2-palmitoylglycerol (2-PG) from Cayman Chemical (Ann Arbor, MI). All reagents and solvents used for extraction and LC/MS were of the highest commercial grade available.

Purified unlabeled N-acylethanolamides (NAEs) and 2-monoacylglycerols (2-MGs) as well as their deuterated analogues were from Cayman Chemical (Ann Arbor, MI) as follows: n-6 arachidonoylethanolamine (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA), n-3 docosahexaenoylethanolamide (DHEA), n-3 eicosapentaenoylethanolamide (EPEA), 2-arachidonoylglycerol (2-AG), 2-oleoylglycerol (2-OG), 2-palmitoylglycerol (2-PG), AEA-d₄, OEA-d₂, PEA-d₄, DHEA-d₄, EPEA-d₄, 2-AG-d₈, and arachidonic acid-d₈ (ARA-d₈, 20:4n-6-d₈. Cis-parinaric acid was from Invitrogen (Grand Island, NY, USA). All reagents and solvents were the highest grade commercially available.